Device and method for treating ophthalmic diseases

ABSTRACT

The invention provides a method for delivering biologically active molecules to the eye by implanting biocompatible capsules containing a cellular source of the biologically active molecule. Also provided is a method of treating ophthalmic diseases using biocompatible capsules.

This application is a continuation of U.S. patent application Ser. No.09/155,066 filed on Sep. 21, 1988 now U.S. Pat. No. 6,297,895, which isa 371 of PCT/US97/04701, filed Mar. 24, 1997 which is a continuation ofU.S. patent application Ser. No. 08/620,982 filed Mar. 22, 1996, NowU.S. Pat. No. 5,904,144, issued May 18, 1999, each of which isincorporated by reference in its entirety.

TECHNICAL FIELD OF THE INVENTION

This invention relates to devices and methods for treatment ofophthalmic diseases and disorders using encapsulated cells forintraocular and periocular delivery of biologically active molecules.

BACKGROUND OF THE INVENTION

There are a number of vision-threatening disorders of the eye for whichthere are presently no good therapies. One major problem in treatment ofsuch diseases is the inability to deliver therapeutic agents into theeye and maintain them there at therapeutically effective concentrations.

Oral ingestion of a drug or injection of a drug at a site other than theeye can provide a drug systemically. However, such systemicadministration does not provide effective levels of the drugspecifically to the eye. In many ophthalmic disorders involving theretina, posterior tract, and optic nerve, adequate levels of drug cannotbe achieved or maintained by oral or parenteral routes ofadministration. Further, repeated administration of the drug may benecessary to achieve these concentrations. However, this may produceundesired systemic toxicity. For example, subcutaneously orintramuscularly administered alpha-interferon in adults may result incomplications such as flu-like symptoms with fatigue, anorexia, nausea,vomiting, thrombocytopenia, and leukopenia.

Ophthalmic conditions have also been treated using drugs applieddirectly to the eye in either liquid or ointment form. This route ofadministration however is only effective in treating problems involvingthe superficial surface of the eye and diseases which involve the corneaand anterior segment of the eye. Topical administration of drugs isineffective in achieving adequate concentrations of drug in the sciera,vitreous, or posterior segment of the eye. In addition, topical eyedrops may drain from the eye through the nasolacrimal duct and into thesystemic circulation, further diluting the medication and riskingunwanted systemic side effects. Furthermore, the drug is administeredindiscriminately to all tissue compartments of the eye, including thosethat may not need the medication and may in fact suffer unwanted sideeffects to the drug.

Delivery of drugs in the form of topical eye drops is also of littleutility when the drug is a protein or peptide that lacks the ability tocross the cornea and be made available to the vitreous, retina, or othersubretinal structures such as the retinal pigment epithelium (“RPE”) orchoroidal vasculature. In addition, many proteins or peptides are highlyunstable and are therefore not easily formulated for topical delivery.

Direct delivery of drugs into the eye by topical insert has also beenattempted. However, this method is not desirable. Topical insertsrequire patient self-administration and thus education on insertion andremoval. This demands a certain degree of manual dexterity, which can beproblematic for geriatric patients. In many instances such inserts maycause eye irritation. These devices are prone to inadvertent loss due tolid laxity. In addition, these devices provide drug only to the corneaand anterior chamber, and do not provide any pharmacologic advantageover eye drops.

Another extraocular insert is a contact lens delivery system thatreleases medication over an extended period. See, e.g., JAMA, 260:24, p.3556 (1988). The lens generally only lasts for a matter of hours or daysbefore dissolving or releasing all of the therapeutic compound.Continuous delivery of medication is inconvenient, requiring frequentre-application. Again, these contact lenses only provide drug to thecornea and anterior chamber.

In rare cases, direct delivery of drugs has also been accomplished usingextemraliz tubes. This requires insertion of one end of a tube into thecorner of the patient's eye. The other end of the tube is taped to thepatient's forehead and terminates in a septum, through which medicationis delivered. This method is undesirable, being both uncomfortable andinconvenient. Since medication must be injected through the septum, thedevice is incapable of continuous delivery of medication. Furthermore,such tubes may become infected and in some cases ultimately threaten thepatient's vision.

Direct delivery of drugs can also be accomplished by the intraocularinjection of the drug, or microspheres that contain the drug. However,microspheres tend to migrate within the eye, either into the visual axisor into adjacent tissue sites.

Most previous intraocular inserts for direct delivery of drugs into theeye have been unsuccessful either because they are unsuitable forlong-term use or are uncomfortable to use. For example, the oculardevice disclosed in U.S. Pat. No. 3,828,777 is not anchored intoposition, thus causing pain, irritation, foreign body sensation, retinaldetachments, and watering when the device moves. Other ocular insertsdisclosed in patents do not disclose sizes or shapes that would allowlong-term retention of the insert. See, e.g., U.S. Pat. No. 4,343,787;U.S. Pat. No. 4,730,013; U.S. Pat. No. 4,164,559. Even in patentsasserting an improved retention and prolonged period of use, thecontemplated period is measured in days, such as 7 to 14 days. See,e.g., U.S. Pat. No. 5,395,618.

One intraocular insert is currently available for delivery ofganciclovir to the eye. Known as Vitrasert, the device consists of anonerodible, polymer-based, sustained-release package containingganciclovir, a non-proteinaceous nucleoside analog. The device issurgically implanted in the vitreous humor of the eye to treatcytomegalovirus retinitis. See, e.g., Anand, R., et al., Arch.Ophthalmol., 111, pp. 223-227 (1993).

Another intraocular insert is disclosed by U.S. Pat. No. 5,466,233. Thistack-shaped device is surgically implanted so that the head of the tackis external to the eye, abutting the scleral surface. The post of thetack crosses the sclera and extends into the vitreous humor, where itprovides for vitreal drug release.

However, release of proteins from such devices (or other erodible ornonerodible polymers) can be sustained for only short periods of timedue to protein instability. Such devices are unsuitable for long-termdelivery of most, if not all, protein molecules.

Clinical treatment for retinal and choroidal neovascularization includesdestruction of new vessels using photocoagulation or cryotherapy.However, side effects are numerous and include failure to controlneovascularization, destruction of macula and central vision, anddecrease in peripheral vision. See, e.g., Aiello, L. P., et al., PNAS,92, pp. 10457-10461 (1995).

A number of growth factors show promise in the treatment of oculardisease. For example, BDNF and CNTF have been shown to slow degenerationof retinal ganglion cells and photoreceptors in various animal models.See, e.g., Genetic Technology News, vol. 13, no. 1 (January 1993). Nervegrowth factor has been shown to enhance retinal ganglion cell survivalafter optic nerve section and has also been shown to promote recovery ofretinal neurons after ischemia. See, e.g., Siliprandi, et al., Invest.Ophthalmol. & Vis. Sci., 34, pp. 3232-3245 (1993).

Direct injection of neurotrophic factors to the vitreous humor of theeye has been shown to promote the survival of retinal neurons andphotoreceptors in a variety of experimentally induced injuries as wellas inherited models of retinal diseases. See, e.g., Faktorovich et al.,Nature, vol. 347 at 83 (Sep. 6, 1990); Siliprandi et al., InvestigativeOphthalmology and Visual Science, 34, p. 3222 (1993); LaVail et al.,PNAS, 89, p. 11249 (1992); Faktorovich et al., Nature, 347, pp. 83-86(1990).

However, previous methods of delivery of such neurotransminers, growthfactors, and neurotrophic factors have significant drawbacks. Someproblems stem from the fact that growth factors do not cross theblood-brain barrier well and are readily degraded in the bloodstream.Further, problems arise with direct injection into the vitreous. Forexample, direct injection of bFGF resulted in an increased incidence ofretinal macrophages and cataracts. See LaVail, PNAS, 89, p. 11249(1992).

Accordingly, delivery of biologically active molecules to the eyewithout adverse effects remains a major challenge.

SUMMARY OF THE INVENTION

This invention provides a novel method of treating ophthalmic diseasesand disorders by intraocular and periocular delivery of acontinuously-produced source of a suitable biologically active molecule(“BAM”).

A capsule containing a cellular source of the BAM is surgically placedin the desired location in the eye.

The capsule jacket comprises a membrane surrounding the encapsulatedcells and interposes a physical barrier between the cells and thepatient. The capsule may be retrieved from the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a horizontal cross section of the eye,indicating a macrocapsule implanted in the vitreous. The diagram is notto scale, and for the sake of clarity shows the capsule in anapproximate placement—when actually placed in the human eye, thepreferred vitreous placement is in the anterior portion of the vitreous.The letter “A” refers to the sciera, “B” refers to Tenon's capsule, and“C” refers to the conjunctiva.

FIG. 2 is a schematic diagram of a side view of the eye showing animplanted capsule beneath Tenon's capsule.

FIG. 3A shows a device with frangible hub assembly for loading.

FIG. 3B depicts the device after detachment of the frangible hub. Thedevice has an eyelet for tethering the device in the eye.

FIG. 4A shows a device with frangible hub assembly for loading.

FIG. 4B depicts the device after detachment of the frangible hub. Thedevice has a disk for tethering the device.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to delivery of biologically active molecules(“BAMs”) intraocularly (e.g., in the anterior chamber, posteriorchamber, or vitreous) or periocularly (e.g., within or beneath Tenon'scapsule), or both. The invention may be useful in providing controlledand sustained release of BAMs effective in treating various ophthalmicdisorders, ophthalmic diseases, or diseases which have ocular effects.

The devices and techniques of this invention provide numerous advantagesover other delivery routes:

Drug can be delivered to the eye directly, reducing or minimizingunwanted peripheral side effects; very small doses of drug (nanogram orlow nicrogram quantities rather than milligrams) can be deliveredcompared with topical applications, also potentially lessening sideeffects; the viable cells of the devices continuously produce newlysynthesized product, avoiding the fluctuation in drug dose thatcharacterizes injection delivery of drugs; and the devices and methodsof this invention are less invasive than many of the prior art devicesand surgical techniques, which result in a large number of retinaldetachments.

Most, if not all, ophthalmic diseases and disorders are associated withone or more of three types of indications: (1) angiogenesis, (2)inflammation, and (3) degeneration. To treat these disorders, thedevices of the present invention permit delivery of anti-angiogenicfactors; anti-inflammatory factors; factors that retard celldegeneration, promote cell sparing, or promote cell growth; andcombinations of the foregoing. Based on the indications of a particulardisorder, one of ordinary skill in the art can administer any suitablemolecule or combination of molecules from the three groups at thedosages specified below.

Diabetic retinopathy, for example, is characterized by angiogenesis.This invention contemplates treating diabetic retinopathy by implantingdevices delivering one or more anti-angiogenic factors eitherintraocularly, preferably in the vitreous, or periocularly, preferablyin the subTenon's region. We most prefer delivery into the vitreous forthis indication. It is also desirable to co-deliver one or moreneurotrophic factors, either intraocularly or periocularly, preferablyintraocularly, and most preferably intravitreally.

Uveitis involves inflammation. This invention contemplates treatinguveitis by intraocular, preferably vitreal or anterior chamber,implantation of devices secreting one or more anti-inflammatory factors.

Retinitis pigmentosa, by comparison, is characterized by retinaldegeneration. This invention contemplates treating retinitis pigmentosaby intraocular, preferably vitreal, placement of devices secreting oneor more neurotrophic factors.

Age-related macular degeneration involves both angiogenesis and retinaldegeneration. This invention contemplates treating this disorder byusing the inventive devices to deliver one or more neurotrophic factorsintraocularly, preferably to the vitreous, and/or one or moreanti-angiogenic factors intraocularly or periocularly, preferablyperiocularly, most preferably to the sub-Tenon's region.

Glaucoma is characterized by increased ocular pressure and loss ofretinal ganglion cells. Treatments for glaucoma contemplated in thisinvention include delivery of one or more neuroprotective agents thatprotect cells from excitotoxic damage. Such agents includeN-methyl-D-aspartate (NMDA) antagonists, cytokines, and neurotrophicfactors, delivered intraocularly, preferably intravitreally.

Any suitable DAM may be delivered according to the devices, systems, andmethods of this invention. Such molecules include neurotransmitters,cytokines, lymphokines, neuroprotective agents, neurotrophic factors,hormones, enzymnes, antibodies, and active fragments thereof Threepreferred types of BAMs are contemplated for delivery using the devicesof the present invention: (1) anti-angiogenic factors, (2)anti-inflammatory factors, and (3) factors that retard celldegeneration, promote cell sparing, or promote cell growth.

The anti-angiogenic factors contemplated for use include vasculostatin,angiostatin, endostatin, anti-integrins, vascular endothelial growthfactor inhibitors (VEGF-inhibitors), platelet factor 4, heparinase, andbFGF-binding molecules. The VEGF receptors Flt and Flk are alsocontemplated. When delivered in the soluble form these molecules competewith the VEGF receptors on vascular endothelial cells to inhibitendothelial cell growth.

VEGF inhibitors may include VEGF-neutralizing chimeric proteins such assoluble VEGF receptors. See Aiello, PNAS, 92, 10457 (1995). Inparticular, they may be VEGF-receptor-IgG chimeric proteins. AnotherVEGF inhibitor contemplated for use in the present invention isantisense phosphorothiotac oligodeoxynucleotides (PS-ODNs).

Intraocularly, preferably in the vitreous, we contemplate delivery of ananti-angiogenic factor in a dosage range of 50 pg to 500 ng, preferably100 pg to 100 ng, and most preferably 1 ng to 50 ng per eye per patientper day. For periocular delivery, preferably in the sub-Tenon's space orregion, slightly higher dosage ranges are contemplated of up to 1 μg perpatient per day.

The anti-inflammatory factors contemplated for use in the presentinvention include antiflammins (see, e.g., U.S. Pat. No. 5,266,562,incorporated herein by reference), beta-interferon (IFN-β),alpha-interferon (IFN-α), TGF-beta, interleukin-10 (IL-10), andglucocorticoids and mineralocorticoids from adrenal cortical cells. Itshould be noted that certain BAMs may have more than one activity. Forexample, it is believed that IFN-α and IFN-β may have activities as bothanti-inflammatory molecules and as anti-angiogenic molecules.

Intraocularly, preferably in the vitreous, we contemplate delivery of ananti-inflammatory factor in a dosage range of 50 pg to 500 ng,preferably 100 pg to 100 ng, and most preferably 1 ng to 50 ng per eyeper patient per day. For periocular delivery, preferably in thesub-Tenon's space or region, slightly higher dosage ranges arecontemplated of up to 1 μg per patient per day.

The factors contemplated for use in retarding cell degeneration,promoting cell sparing, or promoting new cell growth are collectivelyreferred to a herein as “neurotrophic factors”. The neurotrophic factorscontemplated include neurotrophin 4/5 (NT-4/5), cardiotrophin-1 (CT-1),ciliary neurotrophic factor (CNTF), glial cell line derived neurotrophicfactor (GDNF), nerve growth factor (NGF), insulin-like growth factor-1(IGF-1), neurotrophin 3 (NT-3), brain-derived neurotrophic factor(BDNF), PDGF, neurturin, acidic fibroblast growth factor (aFGF), basicfibroblast growth factor (bFGF), EGF, neuregulins, heregulins,TGF-alpha, bone morphogenic proteins (BMP-1, BMP-2, BMP-7, etc.), thehedgehog family (sonic hedgehog, indian hedgehog, and desert hedgehog,etc.), the family of transforming growth factors (including, e.g.,TGFβ-1, TGFβ-2, and TGFβ-3), interleukin 1-B (IL1-β), and such cytokinesas interleukin-6 (IL-6), IL-10, CDF/LIF, and beta-interferon (IFN-β).The preferred neurotrophic factors are GDNF, BDNF, NT-4/5, neurturin,CNTF, and CT-1.

Intraocularly, preferably in the vitreous, we contemplate delivery of aneurotrophic factor in a dosage range of 50 pg to 500 ng, preferably 100pg to 100 ng, and most preferably 1 ng to 50 ng per eye per patient perday. For periocular delivery, preferably in the sub-Tenon's space orregion, slightly higher dosage ranges are contemplated of up to 1 μg perpatient per day.

Modified, truncated, and mutein forms of the above-mentioned moleculesare also contemplated. Further, active fragments of these growth factors(i.e., those fragments of growth factors having biological activitysufficient to achieve a therapeutic effect) are also contemplated. Alsocontemplated are growth factor molecules modified by attachment of oneor more polyethylene glycol (PEG) or other repeating polymeric moieties.Combinations of these proteins and polycistronic versions thereof arealso contemplated.

A gene of interest (i.e., a gene that encodes a suitable BAM) can beinserted into a cloning site of a suitable expression vector by usingstandard techniques. The nucleic acid and amino acid sequences of thehuman (and other mammalian) genes encoding the above identified BAMs areknown. See, e.g., U.S. Pat. Nos. 4,997,929; 5,141,856; 5,364,769;5,453,361; WO 93/06116; WO 95/30686, incorporated herein by reference.

The expression vector containing the gene of interest may then be usedto transfect the desired cell line. Standard transfection techniquessuch as calcium phosphate co-precipitation, DEAE-dextran transfection orelectroporation may be utilized. Commercially available mammaliantransfection kits may be purchased from e.g., Stratagene.Transgenic-mouse-derived cell lines can also be used. See, e.g., Hammanget al., Methods in Neurosci., 21, p. 281 (1994).

A wide variety of host/expression vector combinations may be used toexpress the gene encoding the growth factor, or other BAM(s) ofinterest.

Suitable promoters include, for example. the early and late promoters ofSV40 or adenovirus and other known non-retroviral promoters capable ofcontrolling gene expression.

Useful expression vectors, for example, may consist of segments ofchromosomal, non-chromosomal, and synthetic DNA sequences, such asvarious known derivatives of SV40 and known bacterial plasmids, e.g.,pUC, pBlueScript™ plasmids from E. coli including pBR322, PCR1, pMB9,and their derivatives.

Expression vectors containing the geneticin (G418) or hygromycin drugselection genes (Southern, P. J., In Vitro, 18, p. 315 (1981), Southern,P. J. and Berg, P., J. Mol. Appl. Genet., 1, p. 327 (1982)) are alsouseful. These vectors can employ a variety of differentenhancer/promoter regions to drive the expression of both a biologicgene of interest (e.g., NGF) and/or a gene conferring resistance toselection with toxin such as G418 or hygromycin B. A variety ofdifferent mammalian promoters can be employed to direct the expressionof the genes for G418 and hygromycin B and/or the biologic gene ofinterest.

Examples of expression vectors that can be employed are the commerciallyavailable pRC/CMV, pRC/RSV, and pCDNA1NEO (In Vitrogen).

If cells of a CNS origin are used, preferably the promoter is selectedfrom the following group:

promoters of hDBH (human dopamine beta hydroxylase) (Mercer et al.,Neuron, 7, pp. 703-716, (1991)), hTH (human tyrosine hydroxylase)(Kaneda, et al., Neuron, 6, pp. 583-594 (1991)), hPNMT (humanphenylethanolamine N-methyltransferase) (Baetge et al., PNAS, 85, pp.3648-3652 (1988)), mGFAP (mouse glial fibrillary acidic protein)(Besnard et al., J. Biol. Chem., 266, pp. 18877-18883 (1991)), myelinbasic protein (MBP), mNF-L (mouse neurofilament-light subunit) (Nakahiraet al., J. Biol. Chem., 265, pp. 19786-19791 (1990)), hPo (human P₀, thepromoter for the gene encoding the major myelin glycoprotein in theperipheral nervous system) (Lemke et al., Neuron, 1, pp. 73-83 (1988)),mMt-1 (mouse metallothionein I), rNSE (rat neuron-specific enolase)(Sakimura, et al., Gene, 60, pp. 103-113 (1987)), and the like.

In one preferred embodiment, the phosphoglycerate kinase (PGK) promoteris used. See, e.g., Adra et al., Gene, 60, pp. 65-74 (1987). The pPIvector is one preferred expression vector using the PGK promoter todrive the expression of the gene of interest (i.e. the gene encoding theBAM). This vector also uses the SV40 early promoter to drive expressionof neo phosphotransferase, a selectable marker. One may optimize orenhance expression of a BAM from the pPI vector by inserting the Kozaksequence and/or the Ig signal peptide. The pPI vector also contains amutant DHFR gene suitable for MTX amplification.

In another embodiment, the pNUT expression vector, which contains thecDNA of the mutant DHFR and the entire pUC18 sequence including thepolylinker, can be used. See, e.g., Aebischer, P., et al.,Transplantation, 58, pp. 1275-1277 (1994); Baetge et al., PNAS, 83, pp.5454-58 (1986). The pNUT expression vector can be modified such that theDHFR coding sequence is replaced by the coding sequence for G418 orhygromycin drug resistance. The SV40 promoter within the pNUT expressionvector can also be replaced with any suitable constitutively expressedmammalian promoter, such as those discussed above.

Increased expression can be achieved by increasing or amplifying thecopy number of the transgene encoding the desired molecule, usingamplification methods well known in the art. Such amplification methodsinclude, e.g., DHFR amplification (see, e.g., Kaufman et al., U.S. Pat.No. 4,470,461) or glutamine synthetase (“GS”) amplification (see, e.g.,U.S. Pat. No. 5,122,464, and European published application EP 338,841).

A wide variety of cells may be used. These include well known, publiclyavailable immortalized cell lines as well as dividing primary cellcultures. Examples of suitable publicly available cell lines include,Chinese hamster ovary (CHO), mouse fibroblast (L-M), NIH Swiss mouseembryo (NIH/3T3), African green monkey cell lines (including COS-1,COS-7, BSC-1, BSC40, BMT-10, and Vero), rat adrenal pheochromocytoma(PC12 and PC12A), AT3, rat glial tumor (C6), astrocytes, and otherfibroblast cell lines. Primary cells that may be used includeEGF-responsive neural stem cells and their differentiated progeny(Reynolds and Weiss, Science, 255, pp. 1707-1710 (1992)),bFGF-responsive neural progenitor stem cells derived from the CNS ofmammals (Richards et al., PNAS 89, pp. 8591-8595 (1992), Ray et al.,PNAS 90, pp. 3602-3606 (1993)), CNS neural stem cells that are bothEGF-responsive and bFGF-responsive, primary fibroblasts, Schwann cells,β-TC cells, Hep-G2 cells, oligodendrocytes and their precursors,myoblasts (including L6 and C₂C₁₂ cells), chondrocytes or chondroblasts,and the like.

Conditionally-immortalized cells may also be used. Such cells includecells with temperature sensitive oncogenes, or cells engineered withchimeric genes composed of an oncogene under the direction of aninducible promoter element.

One preferred cell type chosen for the gene transfer technique is thebaby hamster kidney (BHK) cell. BHK cells are particularly amenable toMTX amplification, most likely because they do not express a highlyfunctional DHFR gene.

The suitable cell types include cells from allogeneic and xenogeneicsources. A particular advantage to using xenogeneic cells is that in theunlikely event of membrane or device failure, such cells are more likelyto be targeted for destruction by the immune system.

For delivery in the eye, it may be particularly beneficial to employprimary cells (including primary cells that can be induced to divideusing mitogens such as EGF or bFGF or the like) or cell lines,conditionally-immortalized or otherwise, derived from various regions ofthe eye. Potentially useful cell types include lens epithelial cells,glial and neuronal elements of the neural retina, photoreceptor cells,retinal pigmented epithelial cells, Schwann cells and other ciliary bodycells, and the like. Such cells can be allogeneic or xenogeneic.

As used herein “a biocompatible capsule” means that the capsule, uponimplantation in a host mammal, does not elicit a detrimental hostresponse sufficient to result in the rejection of the capsule or torender it inoperable, for example through degradation.

As used herein “an immunoisolatory capsule” means that the capsule uponimplantation into a mammalian host minimizes the deleterious effects ofthe host's immune system on the cells within its core. To beimmunoisolatory, the capsule should provide a physical barriersufficient to prevent detrimental immunological contact between theisolated cells and the host's immune system. The thickness of thisphysical barrier can vary, but it will always be sufficiently thick toprevent direct contact between the cells and/or substances on eitherside of the barrier. The thickness of this region generally rangesbetween 5 and 200 microns; thicknesses of 10 to 100 microns arepreferred, and thickness of 20 to 75 microns are particularly preferred.

The exclusion of IgG from the core of the vehicle is not the touchstoneof immunoisolation, because in most cases IgG alone is insufficient toproduce cytolysis of the target cells or tissues. Thus, forimmunoisolatory capsules, jacket nominal molecular weight cutoff(MWCO)values between 50-2000 kD are contemplated. Preferably, the MWCO isbetween 50-700 kD. Most preferably, the MWCO is between 70-300 kD. See,e.g., WO 92/19195. If immunoisolation is not required, the jacket can bemicroporous. See, e.g., U.S. Pat. Nos. 4,968,733; 4,976,859; and4,629,563; all incorporated herein by reference.

A variety of biocompatible capsules are suitable for delivery ofmolecules according to this invention. Useful biocompatible polymercapsules comprise (a) a core which contains a cell or cells, eithersuspended in a liquid medium or immobilized within a biocompatiblematrix, and (b) a surrounding jacket comprising a membrane which doesnot contain isolated cells, which is biocompatible, and permitsdiffusion of the cell-produced BAM into the eye.

Many transformed cells or cell lines are advantageously isolated withina capsule having a liquid core, comprising, e.g., a nutrient medium, andoptionally containing a source of additional factors to sustain cellviability and function.

Alternatively, the core may comprise a biocompatible matrix of ahydrogel or other biocompatible matrix material which stabilizes theposition of the cells. The term “hydrogel” herein refers to a threedimensional network of cross-linked hydrophilic polymers. The network isin the form of a gel, substantially composed of water, preferably gelsbeing greater than 90% water.

Any suitable matrix or spacer may be employed within the core, includingprecipitated chitosan, synthetic polymers and polymer blends,microcarriers, and the like, depending upon the growth characteristicsof the cells to be encapsulated. Aternatively, the capsule may have aninternal scaffold. The scaffold may prevent cells from aggregating andimprove cellular distribution within the device. See PCT publication no.WO 96/02646.

Preferably, for implant sites that are not immunologically privileged,such as periocular sites, the capsules are immunoisolatory.

The capsule can be any suitable configuration, including cylindrical,rectangular, disk-shaped, patch-shaped, ovoid, stellate, or spherical.Configurations that tend to lead to migration of the capsules from thesite of implantation, such as spherical, are not preferred. Forimplantations in the vitreous, flat sheets may not be preferred becausethey may block the visual path to the retina.

Preferably the device has a tether that aids in maintaining deviceplacement during implant and aids in retrieval. Such a tether may haveany suitable shape that is adapted to secure the capsule in place. Inone embodiment, the tether is shaped like an eyelet, so that suture maybe used to secure the tether (and thus the capsule) to the sclera, orother suitable ocular structure. In another embodiment, the tether iscontinuous with the capsule at one end, and forms a pre-threaded sutureneedle at the other end. The capsules contemplated here have a minimumcore volume of about 1 to 20 μl, most preferably about 1 to 10 μl.

In a hollow fiber configuration, the fiber will have an inside diameterof less than 1000 microns, preferably less than 950 microns. In oneseries of embodiments, the device is configured to have an 870 μm innerdiameter and a length of about 8.5 mm. In another series of embodiments,the device is configured to have a 500 μm inner diameter and a length of10.5 mm. For implantation in the eye, in a hollow fiber configurationthe capsule will preferably be between 0.4 cm to 1.5 cm in length, mostpreferably between 0.5 to 1.0 cm in length. Longer devices may beaccommodated in the eye, however, a curved or arcuate shape may berequired for secure and appropriate placement. The hollow fiberconfiguration is preferred for intraocular placement.

For periocular placement, either a hollow fiber configuration (withdimensions substantially as above) or a flat sheet configuration iscontemplated. The upper limit contemplated for a flat sheet isapproximately 5 mm×5 mm—assuming a square shape. Other shapes withapproximately the same surface area are also contemplated.

The hydraulic permeability will typically be in the range of 1-100mls/min/M²/mmHg, preferably in the range of 25 to 70 mls/min/M²/mmHg.The glucose mass transfer coefficient of the capsule, defined, measured,and calculated as described by Dionne et al., ASAIO Abstracts, p. 99(1993), and Colton et al., The Kidney, eds., Brenner BM and Rector FC,pp. 2425-89 (1981) will be greater than 10⁻⁶ cm/sec, preferably greaterthan 10⁻⁴ cm/sec.

The capsule jacket may be manufactured from various polymers and polymerblends including polyacrylates (including acrylic copolymers),polyvinyildenes, polyvinyl chloride copolymers, polyurethanes,polystyrenes, polyamides, cellulose acetates, cellulose nitrates,polysulfones (including polyether sulfones), polyphosphazenes,polyacrylonitriles, poly(acrylonitrilelcovinyl chloride), as well asderivatives, copolymers, and mixtures thereof. Capsules manufacturedfrom such materials are described, e.g., in U.S. Pat. Nos. 5,284,761 and5,158,881, incorporated herein by reference. Capsules formed from apolyether sulfone (PES) fiber, such as those described in U.S. Pat. Nos.4,976,859 and 4,968,733, incorporated herein by reference, may also beused.

Depending on the outer surface morphology, capsules have beencategorized as Type 1 (T1), Type 2 (T2), Type 1/2 (T1/2), or Type 4(T4). Such membranes are described, e.g., in Lacy et al., “MaintenanceOf Normoglycemia In Diabetic Mice By Subcutaneous Xenografts OfEncapsulated Islets.” Science, 254, pp. 1782-84 (1991), Dionne et al.,WO 92/19195 and Baetge, WO 95/05452. We prefer a smooth outer surfacemorphology.

Capsule jackets with permselective immunoisolatory membranes arepreferable for sites that are not immunologically privileged. Incontrast, microporous membranes or permselective membranes may besuitable for immunologically privileged sites. For implantation intoimmunologically privileged sites, we prefer capsules made from the PESmembranes.

Any suitable method of sealing the capsules may be used, including theemployment of polymer adhesives and/or crimping, knotting, and heatsealing. These sealing techniques are known in the art. In addition, anysuitable “dry” sealing method can also be used. In such methods, asubstantially non-porous fitting is provided through which thecell-containing solution is introduced. Subsequent to filling, thecapsule is sealed. Such a method is described in copending U.S.application Ser. No. 08/082,407, herein incorporated by reference (seealso PCT/US94/07015). That application describes the frangible hubassembly shown diagrammatically in FIGS. 3 and 4 that can be used toconveniently load and seal the devices of this invention.

We contemplate use of the present invention to treat a wide variety ofophthalmic diseases and disorders characterized by but not limited toangiogenesis, inflammation, degeneration, or some combination thereofSome examples of ophthalmic disorders that may be treated by variousembodiments of the present invention include uveitis, retinitispigmentosa, age-related macular degeneration and other acquireddisorders, retinopathy, retinal vascular diseases and other vascularanomalies, endophthalmitis, infectious diseases, inflammatory butnon-infectious diseases, ocular ischemia syndrome, peripheral retinaldegenerations, retinal degenerations and tumors. choroidal disorders andtumors, vitreous disorders, retinal detachment, non-penetrating andpenetrating trauma, post-cataract complications, and inflammatory opticneuropathies.

Age-related macular degeneration includes but is not limited to dryage-related macular degeneration, exudative age-related maculardegeneration, and myopic degeneration.

Retinopathy includes but is not limited to diabetic retinopathy,proliferative vitreoretinopathy, and toxic retinopathy.

The present invention may be useful for the treatment of ocularneovascularization, a condition associated with many ocular diseases anddisorders and accounting for a majority of severe visual loss. Forexample, we contemplate treatment of retinal ischemia-associated ocularneovascularization, a major cause of blindness in diabetes and manyother diseases; corneal neovascularization, which predisposes patientsto corneal graft failure; and neovascularization associated withdiabetic retinopathy, central retinal vein occlusion, and possiblyage-related macular degeneration.

The present invention may also be used to treat ocular symptomsresulting from diseases or conditions that have both ocular andnon-ocular symptoms. Some examples include AIDS-related disorders suchas cytomegalovirus retinitis and disorders of the vitreous;pregnancy-related disorders such as hypertensive changes in the retina;and ocular effects of various infectious diseases such as tuberculosis,syphilis, lyme disease, parasitic disease, toxocara canis,ophthalmonyiasis, cyst cercosis, and fungal infections.

In one embodiment of the present invention, living cells areencapsulated and surgically inserted (under retrobulbar anesthesia) intothe vitreous of the eye. For vitreal placement, the device may beimplanted through the sclera, with a portion of the device protrudingthrough the sciera. Most preferably, the entire body of the device isimplanted in the vitreous, with no portion of the device protruding intoor through the sciera. Preferably the device is tethered to the sclera(or other suitable ocular structure). The tether may comprise a sutureeyelet (FIG. 3), or disk FIG. 4), or any other suitable anchoring means.The device can remain in the vitreous as long as necessary to achievethe desired prophylaxis or therapy. Such therapies for example includepromotion of neuron or photoreceptor survival or repair, or inhibitionand/or reversal of retinal or choroidal neovascularization, as well asinhibition of uveal, retinal, and optic nerve inflammation. Thisembodiment is preferable for delivering the BAM to the retina.

With vitreal placement, the BAM, preferably a trophic factor, may bedelivered to the retina or the RPE. In addition, retinalneovascularization may be best treated by delivering an anti-angiogenicfactor to the vitreous.

In another embodiment, cell-loaded devices are implanted periocularly,within or beneath the space known as Tenon's capsule. This embodiment isless invasive than implantation into the vitreous and thus is generallypreferred. This route of administration also permits delivery of BAMs(e.g., trophic factors and the like) to the RPE or the retina. Thisembodiment is especially preferred for treating choroidalneovascularization and inflammation of the optic nerve and uveal tract.In general, delivery from this implantation site will permit circulationof the desired BAM to the choroidal vasculature, the retinalvasculature, and the optic nerve.

According to this embodiment we prefer periocular delivery (implantingbeneath Tenon's capsule) of anti-angiogenic molecules, anti-inflammatorymolecules (such as cytokines and hormones), and neurotrophic factors tothe choroidal vasculature to treat macular degeneration (choroidalneovascularization).

Delivery of anti-angiogenic factors directly to the choroidalvasculature (periocularly) or to the vitreous (intraocularly) using thedevices and methods of this invention may reduce the above-mentionedproblems and may permit the treatment of poorly defined or occultchoroidal neovascularization. It may also provide a way of reducing orpreventing recurrent choroidal neovascularization via adjunctive ormaintenance therapy.

In a preferred embodiment, the pNUT vector carrying the desired gene orgenes is transfected into baby hamster kidney (BHK) cells or C₂C₁₂myoblast cells using a standard calcium phosphate transfection procedureand selected with increasing concentrations of methotrexate (1 μM to amaximum of 200 μM) over 8 weeks to produce stable, amplified cell lines.Following this selection, the engineered cells may be maintained invitro in 50-200 μM methotrexate, prior to encapsulation.

The present invention contemplates co-delivery of different factors. Oneof ordinary skill in the art may deliver one or more anti-angiogenicfactors, anti-inflammatory factors or factors retarding celldegeneration, promoting cell sparing, or promoting cell growth,depending on the indications of the particular ophthalmic disorder. Forexample, it may be preferable to deliver one or more neurotrophicfactors together with one or more anti-angiogenic factors, or one ormore anti-inflammatory molecules.

One example is co-delivery of NT-4/5 with endostatin. In this situation,the neurotrophic factor can promote photoreceptor survival while theheparinase would act as an anti-angiogenic factor.

Co-delivery can be accomplished in a number of ways. First, cells may betransfected with separate constructs containing the genes encoding thedescribed molecules. Second, cells may be transfected with a singleconstruct containing two or more genes and the necessary controlelements. We prefer multiple gene expression from a single transcriptover expression from multiple transcription units. See, e.g., Macejak,Nature, 353, pp. 90-94 (1991); WO 94/24870; Mountford and Smith, TrendsGenet., 11, pp. 179-84 (1995); Dirks et al., Gene, 128, pp. 247-49(1993); Martinez-Salas et al., J. Virology, 67, pp. 3748-55 (1993) andMountford et al., Proc. Natl. Acad. Sci. USA, 91, pp. 4303-07 (1994).

Third, either two or more separately engineered cell lines can beco-encapsulated, or more than one device can be implanted at the site ofinterest. And fourth, devices may be implanted in two or more differentsites in the eye concurrently, to deliver the same or different BAMs.For example, it may be desirable to deliver a neurotrophic factor to thevitreous to supply the neural retina (ganglion cells to the RPE) and todeliver an anti-angiogenic factor via the sub-Tenon's space to supplythe choroidal vasculature. While treatment using more than one device iscontemplated and up to five devices per eye, we prefer implantation ofthree devices or less per eye.

Dosage can be varied by any suitable method known in the art. Thisincludes changing (1) the number of cells per device, (2) the number ofdevices per eye, or (3) the level of BAM production per cell. Cellularproduction can be varied by changing, for example, the copy number ofthe gene for the BAM in the transduced cell, or the efficiency of thepromoter driving expression of the BAM. We prefer use of 10³ to 10⁸cells per device, more preferably 5×10⁴ to 5×10⁶ cells per device.

This invention also contemplates use of different cell types during thecourse of the treatment regime. For example, a patient may be implantedwith a capsule device containing a first cell type (e.g., BHK cells). Ifafter time, the patient develops an immune response to that cell type,the capsule can be retrieved, or explanted, and a second capsule can beimplanted containing a second cell type (e.g., CHO cells). In thismanner, continuous provision of the therapeutic molecule is possible,even if the patient develops an immune response to one of theencapsulated cell types.

Alternatively, capsules with a lower MWCO may be used to further preventinteraction of molecules of the patient's immune system with theencapsulated cells.

The methods and devices of this invention are intended for use in aprimate, preferably human host, recipient, patient, subject orindividual.

EXAMPLES Example 1 Preparation and Encapsulation of Cells

BHK-hNGF cells (Winn et al., PNAS, 1994) were produced as follows:

The human NGF (hNGF) gene with the rat insulin intron, as described byHoyle et al., was inserted between the BamHI and SmaI sites of pNUT tobe driven by the metallothionein I promoter. The pNLJT-hNGF constructwas introduced into BHK cells by using a standard calciumphosphate-mediated transfection method. BHK cells were grown inDulbecco's modified Eagle's medium/10% fetal bovineserum/antibiotic/antimycotic/L-glutamine (GIBCO) in 5% CO₂/95% air andat 37° C. Transfected BHK cells were selected in medium containing 200μM methotrexate (Sigma) for 3-4 weeks, and resistant cells weremaintained as a polyclonal population either with or without 200 μMmethotrexate.

The cells were maintained in DMEM with 10% FBS, L-glutamine with 50 μMmethotrexate prior to these experiments. The cells were passaged 1 to 2times per week in the presence of methotrexate. The BHK-hNGF cells andBHK control cells were washed with Hank's buffer, then trypsinized andmixed with Zyderm™ collagen matrix. The cell lines and matrix wereloaded into separate Hamilton syringes that were equipped with blunted,25-gauge needles.

The encapsulation procedure was as follows: The hollow fibers werefabricated from polyether sulfone (PES) with an approximate outsidediameter of 720 μm and a wall thickness of approximately 100 μm(AKZO-Nobel Wüppertal, Germany). These fibers are described in U.S. Pat.Nos. 4,976,859 and 4,968,733, herein incorporated by reference.

The devices comprise:

a semipermeable poly (ether sulfone) hollow fiber membrane fabricated byAKZO Nobel Faser AG;

a hub membrane segment;

a light cured methacrylate (LCM) resin leading end; and

a silicone tether.

The devices had a septal fixture at the proximal end for cellularloading access and were sealed at the distal end. BHK cells wereprepared as a single-cell suspension and infused into the septal port ata density of 15K cells per μl after mixing 1:1 with physiologic collagen(Vitrogen: PC-1). After infusing 1.5 μl of the cellular suspension, theseptum was removed, and the access port was sealed with LCM 23 resin.

The components of the device are commercially available. The LCM glue isavailable from Ablestik Laboratories (Newark, Del.); Luxtrak AdhesivesLCM23 and LCM24).

Example 2 Implantation of Encapsulated Cells into the Sub-Tenon's Space(under Tenon's Capsule)

The patient is prepared and draped in the usual fashion after aretrobulbar injection of 3 cc 2% xylocaine is given to the eye. Aspeculum is inserted beneath the upper and lower lids. The operatingmicroscope is brought into position. A perpendicular incision is madethrough both conjunctiva and Tenon's capsule in the superotemporalquadrant approximately 4 mm back from the limbus. The incision isextended approximately 4-5 mm back from the limbus. At that point, ablunt-tipped scissor is inserted through the incision and is used tobluntly dissect back an additional 5 mm or so on the scleral surface. Atthat point, a membrane device as described in Example 1 is placed inposition through this incision to come to rest on the surface of thesclera. The end of the device that is closest to the limbus has a smallloop that is attached to the cell-loaded device. At this point, a #10-0nylon suture is passed through this loop and sutured into thesuperficial sclera to anchor the membrane to the sclera. At that point,both Tenon's capsule and the conjunctiva are closed with #6-0 plain gutsutures. The speculum is removed and the procedure is concluded.

Example 3 Implantation of Encapsulated Cells into the Vitreous

The patient is prepared and draped in the usual fashion after aretrobulbar injection of 2% xylocaine is given to the eye. At thatpoint, a speculum is inserted into the upper and lower lids and themicroscope is brought into position. A small incision is made throughboth the conjunctiva and Tenon's capsule parallel to and approximately 4mm from the limbus in the supranasal quadrant. The area exposed iscauterized with a wet-field cautery apparatus. A 3 mm incision is thenmade perpendicular to the limbus approximately 4 mm back from thelimbus. The incision is made through the sclera and into the vitreouscavity with a #65 blade. Any of the vitreous which presents itself inthe incision is cut away and removed. At this point, a membrane deviceas described in Example 1 is inserted through the incision into thevitreous cavity. At the end of the membrane, there is a small 2 mm loopthat is attached to the membrane. The loop remains outside the sciera.The sclera is closed with interrupted #9-0 nylon sutures. The #9-0 nylonsutures are also used to anchor this loop of the device to the sclera.The conjunctiva is closed with #60 plain gut sutures.

Example 4 Delivery of Interferon-α (IFN α-2A or IFN α-2B) in theTreatment of Age-Related Macular Degeneration

Candidate cell lines are genetically engineered to express theinterferon molecules. Various interferons may be used; however, weprefer to use IFN α-2A or α-2B. More than one interferon molecule may bedelivered at one time. Various cell lines can also be utilized, weprefer BHK cells.

Cell lines will be encapsulated in preassembled devices substantiallyaccording to example 1. Following the device manufacture, a tether isapplied. This tether contains an eyelet through which suture materialcan be passed. The tether is then used to anchor the device in place toavoid device drift or loss. The cell-loaded devices will be held for astandard period to assure device sterility. The capsule is implantedbeneath the Tenon's capsule according to example 2.

Patients that have been diagnosed with angiographically proven subfovealchoroidal neovascularization involving any part of the foveal avascularzone are to be selected for this therapy.

The effects of IFN α-2a therapy are assessed by visual acuity, clinicalappearance, and fluorescein angiographic appearance. The clinicalappearance of the fundus is assessed subjectively with particularreference to macular elevation by subretinal fluid and the presence ofintraretinal hemorrhage.

Devices will be removed using the same preparation and surgicalprocedure as described above. The device will be placed in vitro andassayed for 24 hours for release of IFN-α. After the assay period, thedevice will be submitted for routine histological analysis to determinethe extent of cell survival.

Example 5 Deliver of HNGF to Neonatal Feline Eyes via EncapsulatedBHK-hNGF Cell Line

BHK-hNGF clone 36 cells were produced according to example 1.

The cells were then encapsulated into 4 mm LCM 24 light-cured capsulesmade from AKZO microporous 10/10 membranes according to example 1. Thecapsules were implanted in neonatal feline eyes substantially accordingto example 4 for 1 month.

Results

In vitro tests for NGF-induced neurite outgrowth were performed beforeand after implantation in the feline eyes. Conditioned medium (CM) fromunencapsulated BHK-control and BHK-hNGF cells was passed through a 0.2μm filter and added to cultures of a PC12 cell subclone, PC12A, grown on6- or 24-well plates at a density of 200,000 cells per ml to test forthe presence of hNGF. Encapsulated cells in the polymeric devices werealso tested for their ability to release bioactive hNGF by placing thedevices in individual wells of a 24-well plate and allowing them toequilibrate for 1-2 days in serum-free defined PC1 medium (Hycor,Portland, Me.); the medium was then removed and replaced with 1 ml offresh PC1 for an additional 24 hour. This CM was collected, placed onthe PC12A cells, and evaluated. Neurite processes that were equal to orgreater than three times the length of the cellbody diameter were scoredas positive. In addition, the rate of neurite induction and thestability of the neurites was examined.

The level of NGF secretion was also tested by ELISA. Quantitation ofhNGF released from both encapsulated and unencapsulated BHK-hNGF cellswas performed by a two-site enzyme immunoassay. The protocol was amodification of that described by Boehringer Mannheim using Nunc-ImmunoMaxisorp ELISA plates. After color development (30 min.), the sampleswere analyzed on a plate reader and measured against recombinant mouseNGF protein standards.

The results were as follows:

ELISA ELISA Capsule Capsule Pre-1* Pre-2* ELISA Post Histology No. BAMpg/24 h. pg/24 hr Explant Cell Survival 1 NGF 152 329 268 (+) 2 NGF 271162 156 (+) 7 Control nd** nd  0 (+) 8 Control nd  nd  0 (+) *Deviceswere assayed twice prior to implantation, once prior to shipment to thecollaborators' laboratory, and a second time immediately prior toimplantation, with a 48-hour time interval between the two assays. #“Pre-1” refers to the results of the first assay, and “Pre-2” refers tothe results of the second assay. **“nd” is an abbreviation for “notdetected.”

In a post explant NGF bioactivity assay, robust neurite outgrowth wasseen for devices 1 and 2 (NGF), but not for devices 3 and 4 (control).

A second similar experiment was conducted. The results are as follows:

ELISA Capsule Capsule ELISA Pre Post Histology No. BAM pg/24 h. ExplantCell Survival  5 NGF 1800  nd* (−)  6 NGF 3900 291 (+) 18 Control nd nd(−) 19 Control nd nd (−) *“nd” is an abbreviation for “not detected.”

In further experiments, BHK cells that secreted hCNTF or NT4/5 wereproduced and encapsulated substantially according to Example 1. However,we experienced difficulties mainly related to shipping and handling ofthese devices, leading to poor cell survival in the capsules. Thus nodata for these capsules is presented here. The shipping difficultiesincluded desiccation, kinking, breakage, and long exposure to lowtemperatures.

Continuing experiments are in progress to deliver various BAMs intonormal and transgenic pig eyes. Pig models are considered one of themost appropriate animal models for the human eye, based on size andvasculature.

We claim:
 1. A method for delivering a biologically active molecule tothe eye comprising: implanting a capsule into the anterior chamber,posterior chamber, or vitreous of the eye, the capsule comprising a corecontaining living cells providing a cellular source of the biologicallyactive molecule and a surrounding biocompatible jacket, the jacketpermitting diffusion of the biologically active molecule into the eye,the capsule configured in a shape adapted to implantation in theanterior chamber, posterior chamber, or vitreous of the eye andoptionally having a tether that is adapted to secure the capsule inplace wherein the dosage of the biologically active molecule thatdiffuses into the eye is between 50 pg and 1000 ng per eye per patientper day.
 2. The method of claim 1 herein the tether is selected from thegroup consisting of a loop, a disk, and a suture eyelet.
 3. The methodof claim 1 wherein the biologically active molecule is ananti-angiogenic factor.
 4. The method of claim 3 wherein theanti-angiogenic factor is selected from the group consisting ofvasculostatin; angiostatin, endostatin, anti-integrins, vascularendothelial growth factor inhibitors (VEGF-inhibitors), platelet factor4, heparinase, bFGF-binding molecules, the VEGF receptor Flt, and theVEGF receptor Flk.
 5. A method for treating ophthalmic disorders in apatient suffering therefrom, comprising implanting into one or both eyesof the patient, a biocompatible capsule, the capsule comprising: (a) acore comprising a cellular source of a biologically active molecule, and(b) a jacket surrounding said core, the jacket comprising abiocompatible material that permits diffusion of the biologically activemolecule to the eye in a therapeutically effective amount, wherein thedosage of the biologically active molecule that diffuses into the eye isbetween 50 pg and 1000 ng per eye per patient per day and wherein theophthalmic disorder is selected from the group consisting of uveitis,retinitis pigmentosa, glaucoma, age-related macular degeneration, anddiabetic retinopathy.
 6. The method of claim 5 wherein the biologicallyactive molecule is an anti-angiogenic factor.
 7. The method of claim 6wherein the anti-angiogenic factor is selected from the group consistingof vasculostatin; angiostatin, endostatin, anti-integrins, vascularendothelial growth factor inhibitors (VEGF-inhibitors), platelet factor4, heparinase, bFGF-binding molecules, the VEGF receptor Flt, and theVEGF receptor Flk.